Caraway Yellows Virus, a Novel Nepovirus from Carum Carvi Yahya Z

Home , Cherry leaf roll virus, Comovirinae, Nepovirus, Secoviridae

Gaafar et al. Virology Journal (2019) 16:70 https://doi.org/10.1186/s12985-019-1181-1

SHORT REPORT Open Access Caraway yellows virus, a novel nepovirus from Carum carvi Yahya Z. A. Gaafar1 , Katja R. Richert-Pöggeler1, Angelika Sieg-Müller1, Petra Lüddecke1, Kerstin Herz1, Jonas Hartrick1, Christina Maaß1, Roswitha Ulrich2 and Heiko Ziebell1*

Abstract A novel nepovirus was identified and characterised from caraway, and tentatively named caraway yellows virus (CawYV). Tubular structures with isomeric virus particles typical for nepoviruses were observed in infected tissues by electron microscopy. The whole genome of CawYV was identified by high throughput sequencing (HTS). It consists of two segments with 8026 nt for RNA1 and 6405 nt for RNA2, excluding the poly(A) tails. CawYV-RNA1 shared closest nt identity to peach rosette mosaic virus (PRMV) with 63%, while RNA2 shared 41.5% with blueberry latent spherical virus (BLSV). The amino acid sequences of the CawYV protease-polymerase (Pro-Pol) and capsid protein (CP) regions share the highest identities with those of the subgroup C nepoviruses. The Pro-Pol region shared highest aa identity with PRMV (80.1%), while the CP region shared 39.6% to soybean latent spherical virus. Phylogenetic analysis of the CawYV-Pro-Pol and -CP aa sequences provided additional evidence of their association with nepoviruses subgroup C. Based on particle morphology, genomic organization and phylogenetic analyses, we propose CawYV as a novel species within the genus Nepovirus subgroup C. Keywords: Caraway, High throughput sequencing, Bipartite genome, Tubular structures, Nepovirus subgroup C

Main text Caraway (Carum carvi L.) is an aromatic biennial Viruses from the genus Nepovirus in the subfamily plant in the Apiaceae family [4]. It is native to Europe, Comovirinae of the Secoviridae family possess a bipartite north Africa and western Asia [5, 6]. Caraway is used as genome consisting of two positive ssRNAs with a 5′ viral a food flavour, fragrance additive, and for medical pur- protein genome-linked (VPg) and a 3′ poly(A) tail [1]. poses as an antibacterial agent with antispasmodic, car- The RNA1 segment encodes the helicase, protease and minative, and appetite stimulant properties [4]. In 2016, its cofactor, replicase and the viral protein genome an organic caraway field showed crop losses. A caraway linked whereas the RNA2 segment encodes the move- plant sample with systemic yellowing was sent to Julius ment and coat proteins [1, 2]. Nepoviruses are the only Kuehn-Institute (JKI) for analysis (Fig. 1a). The sample members of the family Secoviridae known to have a sin- tested positive by DAS-ELISA using antiserum JKI 1283 gle CP [2]. Each of the two RNA segments are encapsi- developed against an uncharacterised nepovirus from dated separately in a non-enveloped icosahedral virion carrot which is likely a strain of cherry leaf roll virus of 25–30 nm in diameter [1]. Nepoviruses can be trans- (CLRV) (unpublished data). The virus was mechanically mitted non-persistently and non-circulatively by nema- transmitted to Nicotiana benthamiana and chlorotic todes, mite and thrips [1, 3]. Seed and pollen local lesions were observed on inoculated leaves transmissions are well-documented [1, 2]. In herbaceous followed by systemic chlorosis and necrosis. Symptom plants, the symptoms induced are often transient with recovery was not observed. The virus was provisionally symptom recovery being a common outcome [2]. named “caraway yellows virus” (CawYV). Electron microscopy (EM) revealed the presence of isomeric virus particles of about 30 nm in diameter in * Correspondence: [email protected] preparations made from the original infected caraway 1Julius Kühn Institute, Institute for Epidemiology and Pathogen Diagnostics, sample (Fig. 1b), indicating the presence of a nepovirus. Messeweg 11-12, 38104 Braunschweig, Germany Additionally, tubules containing virus-like particles in Full list of author information is available at the end of the article

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gaafar et al. Virology Journal (2019) 16:70 Page 2 of 6

Fig. 1 (a) Leaf symptoms of caraway yellows virus (CawYV) on caraway plants; (b) Electron micrograph of CawYV particles from the original infected caraway sample; (c) Electron micrograph showing tubular structure containing virus particles of CawYV in tissue homogenate of infected Nicotiana benthamiana tissue homogenate of N. benthamiana infected with the Geneious assembler (Medium sensitivity/Fast setting). nepovirus were also observed by EM (Fig. 1c). This has 36,634 contigs of lengths between 100 and 23,141 nt also been shown for other nepoviruses e.g., grapevine were generated. A BLASTn search of the contigs against fanleaf virus, where the movement and the capsid pro- a local database for viruses and viroids downloaded from teins act as components of tubular structures (required NCBI showed that two contigs of 7180 and 6341 nt had for cell to cell movement) that traverse the cell wall with 72% identity (73% coverage and zero E-value) to peach the virus particles [1, 7]. rosette mosaic virus (PRMV) and 79% (16% coverage To obtain the full genome of CawYV, double stranded and 1e-90 E-value) to blueberry latent spherical virus RNA (dsRNA) was extracted from infected N. benthami- (BLSV), respectively. The 5′ ends of both RNA segments ana using Double-RNA Viral dsRNA Extraction Mini were confirmed using RNA ligase-mediated amplifica- Kit for Plant Tissue (iNtRON) following the manufac- tion of cDNA ends (RLM-RACE) [8]. The 3′ ends of the turer’s instructions. The extracted dsRNA was sent for two RNA segments were determined by using an oli- library preparation and high throughput sequencing go(d)T primer for cDNA synthesis followed by PCR (HTS) at Eurofins GATC Biotech GmbH. The dsRNA using virus specific primers and the oligo(d)T primer. was fragmented, strand specific cDNA was synthesized The primers used for the 5′ and 3′ ends confirmation using random primers (the dsRNA was denatured at 99 ° are listed in Additional file 1: Table S1. The PCR prod- C for 2 min), followed by adapter ligation and adapter ucts were cloned, sequenced and the resulting sequences specific PCR amplification then sequencing on Illumina were assembled using the map to reference tool and the NovaSeq 6000 platform (2 × 150). original assembled contigs as references. 72,977 of the Using Geneious Prime (v. 2019.0.4), the raw reads (15, quality trimmed reads were assembled to CawYV 468,416) were quality trimmed, filtered, normalized, and complete genome. The assembled genome of CawYV error corrected followed by de novo assembly using was 8026 nt for RNA segment 1 and 6405 nt for RNA Gaafar et al. Virology Journal (2019) 16:70 Page 3 of 6

segment 2 (excluding poly(A) tails). The sequences were Further analyses of the CawYV sequence confirmed its deposited in the GenBank database under accessions identity as a nepovirus. In analogy to other nepoviruses, MK492273 and MK492274. For diagnostic purposes and CawYV RNA1 contains an open reading frame (nt pos- to confirm the presence of CawYV in symptomatic leaf ition 92 to 6733) encoding a polyprotein of 2213 aa in tissue, a primer pair was designed using Primer 3 tool in length. Pairwise comparisons of nt and aa sequences of Geneious (HZ-636 5′ TGA AGA TCC GGG AAA GGC this ORF to its homologues of the other nepoviruses AC 3′ and HZ-637 5′ ACG CTT TCC ACT CTC ACC were performed using ClustalW [10]. The results show TG 3′)[9]. The presence of CawYV was confirmed in that the highest similarity was shared with PRMV at the infected plants by RT-PCR using OneTaq One-Step 66.1% on nt and 68.1% aa levels, respectively (Table 1). RT-PCR Kit (NEB) resulting in amplicons of 481 bp By searching for the different nepovirus motifs using the (data not shown). motif searching tool in Geneious, the locations of the

Table 1 Characteristics and pairwise nucleotide (nt) and amino acid (aa) alignments of the different regions of caraway yellows virus (CawYV) and selected members of subgroup C of the genus Nepovirus i.e., blueberry latent spherical virus (BLSV), blackcurrant reversion virus (BCRV), cherry leaf roll virus (CLRV), grapevine Bulgarian latent virus (GBLV), peach rosette mosaic virus (PRMV), soybean latent spherical virus (SLSV) and tomato ringspot virus (ToRSV) Virus CawYV PRMV BLSV SLSV BCRV GBLV ToRSV CLRV nt length RNA 1 Accession no. MK492273 NC_034214 NC_038764 NC_032270 NC_003509 NC_015492 NC_003840 NC_015414 Complete /−poly(A) 8026 8014 7960 8170 7711 7452 8214 7918 5′UTR 91 41 61 13 66 87 77 11 ORF 6642 6504 6519 6588 6285 6288 6594 6339 3′UTR 1293 1469 1380 1569 1360 1077 1543 1568 RNA 2 Accession no. MK492274 NC_034215 NC_038763 NC_032271 NC_003502 NC_015493 NC_003839 NC_015415 Complete /−poly(A) 6405 5956 6344 5776 6405 5821 7271 6360 5′UTR 94 47 55 23 161 189 75 11 ORF 5022 4425 4896 4197 4881 4500 5649 4770 3′UTR 1289 1484 1393 1556 1363 1132 1547 1579 Pairwise identity % nt % RNA 1 Complete /−poly(A) 63 60.8 49.6 38.5 37 36 33.8 5′UTR 56.1 55.7 30.8 40.9 31.6 52 63.6 ORF 66.1 62.4 53.5 40.4 39.2 36.7 36.3 3′UTR 51.4 53.8 34 40.5 36.5 27 30.9 RNA 2 Complete /−poly(A) 41.3 41.5 37.7 35.9 30.9 38.6 35.6 5′UTR 37.2 30.9 34.8 35.9 36.2 41.3 63.6 ORF 39.9 38.5 39 37 31.8 40.9 37.7 3′UTR 51 55.2 33.4 36.1 37.1 29.4 30.6 aa % RNA 1 ORF 68.1 62.5 48.1 24.5 22.8 23.1 22.6 X1 27.9 29.7 22.9 12.2 10.6 10.8 10.8 X2 Pro-cof 52.9 53.2 33.9 25.8 21.4 15.6 18.5 Hel 82.5 75.6 59.3 28 24.1 22.6 23.2 VPg 75 57.6 56.2 6.1 27.3 25.9 37.9 Pro 79 66.8 51.3 27.6 23.5 24.7 23.1 RdRp 75.5 68.6 53.9 33.8 33.6 33.7 30.9 Pro-Pol 80.1 70.2 54.9 7.1 35.3 36.5 34.6 RNA 2 ORF 22.9 20.1 19 13.1 12.2 22.3 19 HP 23.3 13.1 6.5 7.3 7.6 8.2 7 MP 10 8.4 7.5 8.5 4.1 52.2 54.6 CP 36.5 34.7 39.6 24.3 24.8 26.1 20 Gaafar et al. Virology Journal (2019) 16:70 Page 4 of 6

putative protease cofactor (Pro-cof), the NTP-binding protease-polymerase (Pro-Pol) region aa sequences helicase domain (Hel), the serine protease domain (Pro), showed a closest identity to PRMV Pro-Pol with 80.1% and the RNA-dependent RNA polymerase (RdRp) core (Table 1). domain were found in the RNA1-encoded polyprotein CawYV-RNA2 contains an open reading frame (nt [11]. The putative viral protease cofactor motif position 95 to 5116) encoding a polyprotein of 1673 aa (FX27WX11LX21LXE) was located at aa residues 438– in length. Pairwise comparisons of RNA2-ORF nt and aa 502. The typical NTP-binding helicase motif A sequences to the homologues of other nepoviruses (GX4GKS), motif B (D), and motif C (N) were found at showed the highest similarity with PRMV with 31% nt aa 752–759, 803, and 852, respectively. A serine protease and 21.3% aa identities (Table 1). The conserved move- motif was found at aa 1280–1449 ment protein motif (P) was found at aa position 962 (HX40EX106SGX8GX5GXHX2G) of the CawYV RNA1 [12]. The CP N-terminal five amino acid residues polyprotein sequence (Fig. 2a). The serine at this pos- (SGLEE) together with an alternate capsid protein (CP) ition is unusual for members of the Picornavirales motif (FXFYGWS) were located at aa positions 1119– (where cysteine is usually encoded) but was described 1122 and 1631–1637 [11, 13]. Pairwise analysis of the for some members of genus Nepovirus subgroup C i.e., CP region showed that it shares highest aa identity to BLSV, CLRV, PRMV and soybean latent spherical virus SLSV (39.6%, Table 1). (SLSV). The RNA-dependent RNA polymerase (RdRp) Each of the CawYV polypeptides is predicted to be core domain was located at aa 1774–1880 proteolytically cleaved into putative peptides by the (DX4DX56GX3TX3NX33GDD). Pairwise analysis of the virus-encoded protease. Sequence alignment of all

Fig. 2 (a) Genome organization of CawYV-RNA1 and -RNA2. Each of RNAs 1 and 2 contain a single large open reading frame (in blue). The predicted putative peptides are shown in green, separated by the predicted cleavage site (H/S) (red). The RNA1 polyprotein contains X1 protein, protease cofactor (Pro-cof/X2), helicase (Hel), viral protein genome-linked (VPg), protease (Pro) and RNA-dependent RNA polymerase (RdRP). RNA2 encodes for a polyprotein with hypothetical protein (HP), movement protein (MP) and capsid protein (CP). The conserved nepovirus sequences (domains and motifs) are shown in violet. The protease-polymerase (Pro-Pol) region of CawYV-RNA1 starts with the serine (S) of the protease motif and ends with the (GDD) motif of the polymerase (shown in gold). (b) Maximum-likelihood (ML) phylogenetic trees showing the relationships between CawYV and members of the genus Nepovirus based on aa alignments of the Pro-Pol region and (c) the capsid protein (CP) region. Numbers on branches indicate the bootstrap percentages (1000 replicates, ≥50% are shown). Tomato torrado virus (genus Torradovirus, family Secoviridae) is an outgroup Gaafar et al. Virology Journal (2019) 16:70 Page 5 of 6

nepovirus ORF aa sequences suggest a putative proteo- genus Nepovirus. Further studies are needed to inves- lytic cleavage sites at dipeptides (H/S). This potential tigate the natural mode of transmission and the bio- cleavage site was not identified before in the Secoviridae logical characteristics of CawYV. members. The conserved histidine in the substrate- binding pocket of the protease is known for members of the subgroup C, however the known cleavage sites are Additional file Q/G, Q/S or D/S (confirmed experimentally) [1]. The H/S dipeptide is also found in SLSV, BLSV and PRMV. Additional file 1: Table S1. List of the primers used for caraway yellows ′ ′ Although the VPg motif was not confirmed in the poly- virus 5 and 3 ends confirmation. (DOCX 18 kb) protein of RNA1, the location of the putative VPg do- Abbreviations main could be determined by the H/S dipeptides aa: amino acid; BCRV: blackcurrant reversion virus; BLSV: blueberry latent between the NTP-binding helicase and the protease spherical virus; BRSV: beet ringspot virus; CawYV: caraway yellows virus; using sequence alignment (Fig. 2a). Additionally, the X1 CLRV: cherry leaf roll virus; CP: capsid protein; DAS-ELISA: double antibody sandwich enzyme-linked immunosorbent assay; dsRNA: double stranded putative protein was identified at the N terminus of RNA; EM: electron microscopy; GBLV: grapevine Bulgarian latent virus; RNA1 polyprotein by the presence of a H/S dipeptide Hel: helicase; HTS: high throughput sequencing; ICTV: International potential cleavage site before the protease cofactor motif Committee on Taxonomy of Viruses; JKI: Julius Kuehn-Institute; kDa: kilo ′ Dalton; nm: nanometer; nt: nucleotide; PRMV: peach rosette mosaic virus; of X2 (Fig. 2a). The 5 untranslated regions (UTR) of Pro: protease; Pro-cof: protease cofactor; Pro-Pol: protease-polymerase; the two RNAs were 91 nt for RNA1 and 94 nt for RNA2 RdRp: RNA-dependent RNA polymerase; RLM-RACE: RNA ligase-mediated and shared 61.3% nt identity to each other. The 3’UTRs amplification of cDNA ends; RT-PCR: reverse transcription polymerase chain reaction; SLSV: soybean latent spherical virus; TBRV: tomato black ring virus; (1293 and 1289 nt for RNA 1 and 2 respectively, exclud- ToRSV: tomato ringspot virus; UTR: untranslated regions; VPg: protein ing the poly(A) tail) are 98.4% identical. genome-linked A maximum likelihood tree using MEGA7 software (v 7.0.26) based on the aa alignments of the Pro-Pol and Acknowledgments The authors are grateful to the reviewers for their suggestions. the CP regions were additional evidence for the related- ness of CawYV to the Nepovirus subgroup C (Fig. 2b Authors’ contributions and c) [14]. YZAG and HZ conceived and designed the study; YZAG carried out molecular studies; KR-P performed the electron microscopy experiments; The International Committee on Taxonomy of Vi- YZAG analysed the data; YZAG drafted the manuscript; AS, PL, KH, JH and ruses (ICTV) suggests the following criteria for spe- CM provided technical assistance; RU provided the original sample; Funding cies demarcation [1]: distinct host range; distinct was acquired by HZ; All authors read and approved the final version. vector specificity; absence of cross-protection; differ- Funding ences in antigenic reactions; absence of reassortment This research was financed by the EUPHRESCO project (2015-F-172; The between RNA1 and RNA2; Pro-Pol region aa < 80% application of Next-Generation Sequencing technology for the detection and diagnosis of non-culturable organism: Viruses and viroids). Yahya Z. A. Gaafar and CP region aa < 75% identities. Although the host was funded by the German Egyptian research long term scholarship. Open range was not studied, the closest relatives of CawYV, access publication was enabled by JKI core funding. i.e., PRMV and BLSV, are not known to infect members of the Apiaceae family. The serological cross- Availability of data and materials The datasets used and/or analysed during the current study are available reactivity is well known for members of the same from the corresponding author on reasonable request. genus in the family Secoviridae [1]. This might ex- plain why our antiserum raised against an uncharac- Ethics approval and consent to participate This research does not contain any studies on human or animals. terised strain of CLRV reacted with CawYV. Further investigations are necessary to test the antiserum Consent for publication against other nepoviruses, and attempts are currently All authors agreed to the publication of this manuscript. underway to develop a CawYV-specific antiserum. Competing interests When compared to the closest relative PRMV, the The authors declare no competing interests. Pro-Pol region of CawYV is slightly above the species demarcation limit by 0.1%. However, this was also ob- Author details 1Julius Kühn Institute, Institute for Epidemiology and Pathogen Diagnostics, served for other nepoviruses e.g., beet ringspot virus Messeweg 11-12, 38104 Braunschweig, Germany. 2Regierungspräsidium (BRSV) and tomato black ring virus (TBRV) that Gießen, Schanzenfeldstrasse 8, 325578 Wetzlar, Germany. share 89% aa identity at the Pro-Pol [1] but are yet Received: 26 March 2019 Accepted: 16 May 2019 classified as distinct species. However, the caraway virus-CP region is very different to other nepoviruses sharing only 39.6% aa identity with SLSV. Based on References 1. Thompson JR, Dasgupta I, Fuchs M, Iwanami T, Karasev AV, Petrzik K, et al. these results, we propose the assignment of CawYV ICTV virus taxonomy profile: Secoviridae. J Gen Virol. 2017;98:529–31. https:// as a new virus species within the subgroup C of the doi.org/10.1099/jgv.0.000779. Gaafar et al. Virology Journal (2019) 16:70 Page 6 of 6

2. Fuchs M, Schmitt-Keichinger C, Sanfaçon H. Chapter two - a renaissance in nepovirus research provides new insights into their molecular interface with hosts and vectors. In: Kielian M, Thomas CM, Marilyn JR, editors. Adv Virus Res. Saint Louis: Elsevier Science; 2017. p. 61–105. https://doi.org/10.1016/bs. aivir.2016.08.009. 3. Bergeson GB, Athow KL, Laviolette FA, Thomasine M. Transmission, movement, and vector relationships of tobacco ringspot virus in soybean. Phytopathology. 1964;54:723–8. 4. Iacobellis NS, Lo Cantore P, Capasso F, Senatore F. Antibacterial activity of Cuminum cyminum L. and Carum carvi L. essential oils. J Agric Food Chem. 2005;53:57–61. https://doi.org/10.1021/jf0487351. 5. Bailer J, Aichinger T, Hackl G, de HK, Dachler M. Essential oil content and composition in commercially available dill cultivars in comparison to caraway. Ind Crop Prod. 2001;14:229–39. https://doi.org/10.1016/S0926- 6690(01)00088-7 . 6. Eddouks M, Lemhadri A, Michel J-B. Caraway and caper: potential anti- hyperglycaemic plants in diabetic rats. J Ethnopharmacol. 2004;94:143–8. https://doi.org/10.1016/j.jep.2004.05.006 . 7. Laporte C, Vetter G, Loudes A-M, Robinson DG, Hillmer S, Stussi-Garaud C, Ritzenthaler C. Involvement of the secretory pathway and the cytoskeleton in intracellular targeting and tubule assembly of Grapevine fanleaf virus movement protein in tobacco BY-2 cells. Plant Cell. 2003;15:2058–75. https://doi.org/10.1105/tpc.013896. 8. Coutts RHA, Livieratos IC. A rapid method for sequencing the 5′-and3′-termini of double-stranded RNA viral templates using RLM-RACE. J Phytopathol. 2003; 151:525–7. https://doi.org/10.1046/j.1439-0434.2003.00755.x . 9. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG. Primer3--new capabilities and interfaces. Nucleic Acids Res. 2012;40: e115. https://doi.org/10.1093/nar/gks596 . 10. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23:2947–8. https://doi.org/10.1093/bioinformatics/btm404 . 11. Isogai M, Tatuto N, Ujiie C, Watanabe M, Yoshikawa N. Identification and characterization of blueberry latent spherical virus, a new member of subgroup C in the genus Nepovirus. Arch Virol. 2012;157:297–303. https:// doi.org/10.1007/s00705-011-1177-7. 12. Mushegian AR. The putative movement domain encoded by nepovirus RNA-2 is conserved in all sequenced nepoviruses. Arch Virol. 1994;135:437– 41. https://doi.org/10.1007/BF01310028 . 13. Le Gall O, Candresse T, Dunez J. A multiple alignment of the capsid protein sequences of nepoviruses and comoviruses suggests a common structure. Arch Virol. 1995;140:2041–53. https://doi.org/10.1007/BF01322691 . 14. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33:1870–4. https://doi.org/10.1093/molbev/msw054 .

Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.